Probe density self-considerations and elongation of complementary looped probes where probes are attached to a solid phase

ABSTRACT

In a multiplexed assay method carried out in solution, wherein the solution contains nucleic acid targets and, wherein several different types of oligonucleotide probes, each type having a different sequence in a region designated as a target binding domain, are used to detect the nucleic acid targets, said assay method including a method for increasing the effective concentration of the nucleic acid targets at the surface of a bead to which the oligonucleotide probes are bound, by one or more of the following steps: 
     adjusting assay conditions so as to increase the effective concentration of the targets available for binding to the probes, by one or more of the following: (i) selecting a particular probe density on the surface of the bead; (ii) selecting a solution having an ionic strength greater than a threshold; (ii) selecting a target domain of a size less than a threshold; or (iii) selecting target domains within a specified proximity to a terminal end of the targets.

BACKGROUND

Molecular Stringency in Multiplexed Assays—A self-complementaryoligonucleotide capture probe in a “looped” configuration may be used toadjust molecular stringency in an assay. Assay stringency relates to thepositive results produced by an assay, such that high stringencyconditions generate relatively fewer positive results than lowerstringency conditions. Looped probes are described in WO 01/98765,entitled: “Multianalyte Molecular Analysis Using Application-SpecificRandom Particle Arrays” and U.S. Pat. No. 6,361,945 (assigned to GenProbe, Inc.). Such a probe consists of a 5′-terminal subsequence and acomplementary 3′-terminal subsequence, tethered by an unrelatedsubsequence, the two terminal subsequences capable of forming a duplex(“stem”), and the tether forming a loop, and either the 5′-terminalsubsequence of the 3′-terminal subsequence capable of forming a duplexwith a target nucleic acid. The probe may be attached to a solid phasesuch as an encoded microparticle (“bead”), by way of an appropriatefunctional modification of the 5′terminal subsequence or the loopsubsequence.

Using a fluorescence acceptor and a proximal fluorescence quencher (asdiscussed in U.S. Pat. No. 6,534,274), capture of a target nucleic acidis detected by way of detecting a transition from the Closed (“C”) stateof the capture probe to the Open (“O”) state or the target-associated(“OT”) state, the O-state contributing to “background” fluorescence,independent of target concentration (FIG. 1). In this competitiveequilibrium, low stringency, favoring the closed state, will reduce thelikelihood of formation of the open (or other intermediate state, seeDetailed Description, below) required for probe-target duplex formation,thereby diminishing the detection sensitivity. Conversely, highstringency, favoring the open state, will likewise reduce the likelihoodof target capture—by reducing the stability of any probe-targetduplex—while producing indiscriminate fluorescence, independent ofcaptured target, thereby reducing specificity.

Thus, the use of a looped probe calls for resolution of the conflictbetween detection sensitivity and specificity, preferably by operatingnear an optimal stringency, determined by a choice of buffer conditionsand operating temperature. For typical buffer conditions, whichgenerally are of low ionic strength, e.g. corresponding to saltconcentrations of 50 mM, this step requires selection of an optimaldetection temperature, preferably at or above the range of the midpointof the melting curve where specificity is optimal. Optimal stringenciesgenerally will depend on capture probe sequences, and on targetconfiguration and/or length. Thus, identifying the optimal stringencyrange in a multiplexed assay thus becomes increasingly difficult witheach different probe added, given the dispersion of the melting curveprofiles of a set of different probe-target complexes under given assayconditions.

SUMMARY OF THE INVENTION

Disclosed are methods of enhancing detection sensitivity and expandingthe range of stringencies compatible with detection of specific targets,especially where there is a low target concentration, as typicallyencountered in, e.g., the detection of genomic material from infectiousagents (see e.g., Chen, Martinez & Mulchandani, “Molecular Beacons: AReal-Time Polymerase Chain Reaction Assay for Detecting Salmonella,”Analytical Biochemistry 280, 166-172 (2000)). Also disclosed is a methodof enhancing detection sensitivity by providing for target capture to aself complementary (“looped”) probe, anchored, preferably by its loopsubsequence, at a lateral density of at least a certain preset minimum,on a solid phase carrier, preferably a microparticle (“bead”).

Further disclosed is a method of stabilizing a probe-target complexunder conditions of high stringency by providing for target-mediated,enzyme-catalyzed elongation of the 3′-terminal probe subsequence toconvert the probe-target complex (“OT”), formed as a result of targetcapture and characterized by fluorescence, into an elongation product(“eOT”) of enhanced thermodynamic stability (FIG. 2). The formation ofthe eOT state can be detected by temperature cycling: the eOT complexmay be exposed to higher temperatures without loss of fluorescence—whichwould otherwise result, for a non-elongated complex (in the OT state),from the release of the target at the higher temperature and formationof the closed (“C”) state of the probe—upon subsequent return to lowertemperature.

The formation of this elongation product has at least a three-foldbenefit:

(i) enhance the sensitivity of target detection—by converting the Cstate of the probe into the eOT state; even under conditions of extremestringency, selected, for example, to ensure enzymatic efficiencyparticularly in homogeneous assay designs ((see e.g. “TranscriptionAmplification System with Integrated Multiplex Detection; FunctionalIntegration of Capture, Amplification and Multiplex Detection” filedSep. 2, 2005; Ser. No. 11/218,838, incorporated by reference), thisconversion ensures high detection sensitivity by accumulation ofelongation product, over an extended period of time, by way of randomfluctuations of the closed into the open (or related reactiveintermediate, see below) state permitting target capture andenzyme-catalyzed elongation; to the extent that the eOT state isirreversible under prevailing assay conditions, this conversion is akinto a digital “ON” signal;(ii) enhance the range of optimal stringency of a multiplexedassay—essentially by raising melting temperatures and thereby avoidingoperation in the range of temperatures coinciding with dispersion in themelting curves of multiple distinct probe-target pairs; and(iii) enable the application of allele-specific detection andimplementation of a phasing strategy, in analogy to the phasing methoddescribed in U.S. patent application Ser. No. 10/271,602, entitled:“Multiplexed Analysis of Polymorphic Loci by Concurrent

Interrogation and Enzyme-Mediated Detection,” incorporated by reference.

DESCRIPTION OF FIGURES

FIG. 1 is an illustration showing the closed (“C”), open (“O”), andtarget-associated (“OT”) states of a self-complementary (“looped”)capture probe.

FIG. 2A is an illustration showing the target-mediated, enzymaticelongation of a looped probe labeled with a fluorescence donor on the5′-terminal subsequence and an acceptor on the 3′-terminal subsequence.

FIG. 2B is an illustration showing the target-mediated, enzymaticelongation of non-labeled looped probe.

FIG. 3 is an illustration comparing a volume element of solutioncontaining uniformly distributed capture probes, and a volume elementcontaining a microparticle and capture probes confined to a shell.

FIG. 4A is an illustration showing the configuration of a homogenousassay performed using looped probes displayed on a pre-assembled randomarray of encoded beads.

FIG. 4B is an illustration showing an arrangement for performing ahomogenous assay using looped probes displayed on a pre-assembled randomarray of encoded beads, where the array is mounted on an insert at thetip of a reaction tube and imaged in an inverted imaging arrangement.

FIG. 5 is a representation showing the capture of RNA target tobead-displayed self-complementary capture probes in homogeneous BeadChipassays.

FIG. 6 is an illustration of target “hopping” process and the escapeprocess with concomitant shape relaxation.

FIG. 7A is an illustration of the effect of probe elongation on themelting curves of several probe-target complexes, and FIG. 7B is anillustration of the effect of randomly aborted probe elongation on thedistribution of affinity constants.

FIG. 8 is an illustration of phasing, performed by elongation ofallele-specific looped probes.

FIG. 9 is an illustration showing the configuration of a homogeneousassay performed using with labeled looped probes displayed on encodedsuspended beads.

FIG. 10 is an illustration showing the configuration of a homogeneousassay performed using with non-labeled looped probes displayed onencoded suspended beads.

FIG. 11 is an illustration of into an incubation chamber in place on asilicon wafer.

FIG. 12A is an illustration of a magnetic trap.

FIG. 12B shows the computed field distribution of quantities relevant tomagneto-phoresis.

FIG. 13 shows bead-map plotted with Cy3 against blue, showing threeclusters of beads.

FIG. 14 shows the dose response of target interaction with specific andnon-specific looped probes.

DETAILED DESCRIPTION 1—Mathematical Description of Molecular Stringency:Competitive Target Capture

In general, the interaction of a looped probe with a target nucleic acidwill be governed by a set of coupled equilibria between thenon-fluorescent closed (“C”) state, and the fluorescent open (“O”) stateand the fluorescent target-associated (“OT”) state. Capture of a targetnucleic acid is detected by way of detecting a transition from the C tothe OT state. The 0 state, which is not associated with the target,contributes to a “background fluorescence”. The equations below describemathematically the corresponding coupled equilibria. The four inputparameters are the initial looped probe concentration [P]⁰, initialtarget concentration [T]⁰, and the relevant equilibrium constants.

In the most general situation, the target is permitted to interact notonly with the open but also directly with the closed state of the loopedprobe (in a displacement reaction) so as to form a probe-target complex.For molecular beacon probes in solution—beacons, in contrast to thelooped probes considered here, are designed to form a probe-targetcomplex by way of the loop sequence and thus do not impose molecularstringency—Bonnet et al. reported a mathematical model applicable underconditions of excess target (see Bonnet et al, Proc. Natl. Acad. Sci.USA Vol. 96, pp. 6171-6176, May 1999, Biophysics). Here, we consider themore general situation, i.e., that there is usually low concentration oftarget and excess probe, in assays using solid phase-immobilized probesto detect targets in solution.

Consider first looped probes, exposed to targets, the probes capable ofadopting one of three states: (i) a duplex state (associated withtarget), (ii) a closed state (the complementary stem subsequencesforming a duplex), and (iii) an open state, for example in the form ofan open random coil (prevalent, for example, at high temperature). Atequilibrium:

${\underset{\uparrow \underset{\_}{\mspace{104mu} K_{o}\mspace{101mu}} \uparrow}{{OT}\overset{K_{c}}{\leftrightarrow}{C + T}\overset{K_{co}}{\leftrightarrow}O} + T},$

where OT is the looped probe-target duplex, C is the probe in its closedstate, O is the probe in the form of a random coil, and T is the freetarget. The normalized fluorescence at a given temperature should be thesum of the contribution from each of the three states:

$F = {{\alpha \frac{\lbrack{OT}\rbrack}{P^{0}}} + {\beta \frac{\lbrack C\rbrack}{P^{0}}} + {\gamma \frac{\lbrack O\rbrack}{P^{0}}}}$

where α, β, and γ are the fluorescence quantum efficiency (QE) of thelooped probe in each state, and

P ⁰ =[OT]+[C]+[O]

T ⁰ =[OT]+[T]

The law of mass action gives the following expression for theequilibrium constants governing the dissociation of the looped probe:

${K_{c} = \frac{\lbrack C\rbrack \cdot \lbrack T\rbrack}{\lbrack{OT}\rbrack}},{K_{co} = \frac{\lbrack O\rbrack}{\lbrack C\rbrack}},{K_{o} = {\frac{\lbrack O\rbrack \cdot \lbrack T\rbrack}{\lbrack{OT}\rbrack}.}}$

These affinity constants are related by the following relation:

$K_{co} = {\frac{K_{o}}{K_{c}}.}$

Two limiting cases of interest are:

Excess Probe, i.e., P⁰>>T⁰:

The fraction of probes in each state can be expressed in terms of theequilibrium constants, K_(c) and K_(o) as follows:

$\frac{\lbrack{OT}\rbrack}{P^{0}} = {T^{0}\left( {P^{0} + K_{c} + K_{o}} \right)}^{- 1}$$\frac{\lbrack C\rbrack}{P^{0}} = {K_{c}\left( {K_{c} + K_{o}} \right)}^{- 1}$$\frac{\lbrack O\rbrack}{P^{0}} = {K_{o}\left( {K_{c} + K_{o}} \right)}^{- 1}$

Thus, the total fluorescence intensity is:

F=αT ⁰(P ⁰ ±K _(c) +K _(o))⁻¹ +βK _(c)(K _(c) +K _(o))⁻¹ +γK _(o)(K _(c)+K _(o))⁻¹.

Excess Target, i.e., P⁰<<T⁰:

The fraction of probes in each state again can be expressed in terms ofthe equilibrium constants, K_(c) and K_(o) as follows:

$\frac{\lbrack{OT}\rbrack}{P^{0}} = {T^{0}\left( {T^{0} + K_{c} + K_{o}} \right)}^{- 1}$$\frac{\lbrack C\rbrack}{P^{0}} = {K_{c}\left( {T^{0} + K_{c} + K_{o}} \right)}^{- 1}$$\frac{\lbrack O\rbrack}{P^{0}} = {K_{o}\left( {T^{0} + K_{c} + K_{o}} \right)}^{- 1}$

Thus, the fluorescence intensity is:

F=[αT ⁰ +βK _(c) +γK _(o)](T ⁰ +K _(c) +K _(o))⁻¹.

These equations may be simplified by assuming equality of quantumefficiencies (QE) in the duplex and open states, i.e., α˜γ, andnegligible QE in the closed state, i.e., β˜0:

$\begin{matrix}{\lbrack{OT}\rbrack = \frac{\left( {K_{c} + K_{o}} \right) + P^{0} + {T^{0} \pm \sqrt{\begin{matrix}{\left( {K_{c} + K_{o} + P^{0} + T^{0}} \right)^{2} -} \\{4P^{0}T^{0}}\end{matrix}}}}{2}} & (1)\end{matrix}$

Then, for the case of excess probe, i.e., T⁰<<P⁰:

$\lbrack{OT}\rbrack = \frac{\left( {K_{c} + K_{o}} \right)^{- 1}P^{0}T^{0}}{\left( {1 + {\left( {K_{c} + K_{o}} \right)^{- 1}P^{0}}} \right)}$

and similarly, for excess target, i.e. P⁰<<T⁰:

$\lbrack{OT}\rbrack = \frac{\left( {K_{c} + K_{o}} \right)^{- 1}P^{0}T^{0}}{\left( {1 + {\left( {K_{c} + K_{o}} \right)^{- 1}T^{0}}} \right)}$

Both expressions are equivalent to a Langmuir adsorption isothermdescribing the capture of target to a probe-decorated solid phase in aprocess governed by a single effective affinity constant,K_(eff)=(K_(c)+K_(o))⁻¹

The fraction of signal originating from the probe-target complex,compared to that originating from the open state of the probe, is givenby:

$\eta = {\frac{\lbrack{OT}\rbrack}{\lbrack O\rbrack} = {\frac{\left( {K_{c} + K_{o}} \right)}{K_{o}}\left\{ \frac{\lbrack{OT}\rbrack}{P^{0} - \lbrack{OT}\rbrack} \right\}}}$

Simplified Model: No Displacement—A similar result also is obtained byconsidering the target to interact only with the open form of the loopedprobe in accordance with a coupled equilibrium:

$C\overset{K_{1}}{\Leftrightarrow}{O + T}\overset{K_{2}}{\Leftrightarrow}{OT}$

where K₁ and K₂ are the association equilibrium constants, namely:

$K_{2} = \frac{\lbrack{OT}\rbrack}{\left( {T^{0} - \lbrack{OT}\rbrack} \right)\lbrack O\rbrack}$${or},{\lbrack{OT}\rbrack = \frac{K_{2}{T^{0}\lbrack O\rbrack}}{1 + {K_{2}\lbrack O\rbrack}}}$Similarly$K_{1} = \frac{\lbrack O\rbrack}{\left( {P^{0} - \lbrack O\rbrack - \lbrack{OT}\rbrack} \right)}$${or},{\lbrack O\rbrack = {\frac{K_{1}\left( {T^{0} - \lbrack{OT}\rbrack} \right)}{\left( {1 + K_{1}} \right)} = {\delta \left( {T^{0} - \lbrack{OT}\rbrack} \right)}}}$where $\delta = \frac{K_{1}}{\left( {1 + K_{1}} \right)}$

These two algebraic equations yield:

$\begin{matrix}{{{K_{2}{\delta \lbrack{OT}\rbrack}^{2}} - {\left( {1 + {K_{2}\delta \; T^{0}} + {K_{2}\delta \; P^{0}}} \right)\lbrack{OT}\rbrack} + {K_{2}\delta \; T^{0}P^{0}}} = {{0\lbrack{OT}\rbrack} = {{\frac{\begin{matrix}{\left( {1 + {K_{2}\delta \; T^{0}} + {K_{2}\delta \; P^{0}}} \right) \pm} \\{\sqrt{\left( {1 + {K_{2}\delta \; T^{0}} + {K_{2}\delta \; P^{0}}} \right)^{2} - 4}\left( {K_{2}\delta} \right)^{2}P^{0}T^{0}}\end{matrix}}{2K_{2}\delta}\lbrack{OT}\rbrack} = \frac{\left( {\frac{1}{K_{2}\delta} + T^{0} + P^{0}} \right) \pm \sqrt{\left( {\frac{1}{K_{2}\delta} + T^{0} + P^{0}} \right)^{2} - {4T^{0}P^{0}}}}{2}}}} & (2)\end{matrix}$

Then, for excess probe, i.e., P⁰>>T⁰:

$\lbrack{OT}\rbrack = \frac{\delta \; K_{2}P^{0}T^{0}}{\left( {1 + {\delta \; K_{2}P^{0}}} \right)}$

and similarly, for excess target, i.e. P⁰<<T⁰:

$\lbrack{OT}\rbrack = \frac{\delta \; K_{2}P^{0}T^{0}}{\left( {1 + {\delta \; K_{2}T^{0}}} \right)}$

Both expressions are equivalent to a Langmuir adsorption isothermdescribing the capture of target to a probe-decorated solid phase in aprocess governed by a single effective affinity constant, K_(eff)=δK₂

The fraction of signal originating from the probe-target complex,compared to that originating from the open state of the probe is givenby:

$\eta = {\frac{\lbrack{OT}\rbrack}{\lbrack O\rbrack} = {\delta \left\{ \frac{\lbrack{OT}\rbrack}{P^{0} - \lbrack{OT}\rbrack} \right\}}}$

Both models thus generate similar mathematical expressions for [OT],namely:

$\lbrack{OT}\rbrack = {\frac{1}{2}\left\{ {\left( {K_{eff} + T^{0} + P^{0}} \right) - \sqrt{\left( {K_{eff} + T^{0} + P^{0}} \right)^{2} - {4T^{0}P^{0}}}} \right\}}$

where K_(eff) represents an association equilibrium constant governingthe reaction P+T

OT, between any of the states of the probe, P, and the target-associatedstate, and P⁰ and T⁰ respectively denote the initial concentrations ofthe probe and target. For the general model, K_(eff)=(K_(c)+K_(o))⁻¹ andfor the simplified model, K_(eff)=K₁K₂/(1+K₁).

Both models likewise generate similar expressions for the parameter η,namely:

$\eta = {\lambda \left\{ \frac{\lbrack{OT}\rbrack}{P^{0} - \lbrack{OT}\rbrack} \right\}}$

where λ, for the general model, is given by: λ=(1+K_(c)/K_(o)), and forthe simplified model is given by λ=(1+K_(l))/K_(l).

Under conditions of low coverage, [OT]/P⁰<<1, η increases linearly with[OT] which, in this regime, is in turn linearly dependent on K_(eff).Hence, in this low coverage regime, an increase in K_(eff), reflectingchoice of ionic strength and/or temperature, will lead to an increase η,and hence detection sensitivity. This can be brought about by a choicein buffer conditions such that affinity K_(l) or K_(co) decreases, whichdestabilizes the O state in favor of the OT state.

Probability of Target-Probe Encounter: Solution vs Solid Phase—For giventarget concentration, the probability of a target molecule encounteringa probe is determined by the effective concentration of probes. Withreference to FIG. 3, consider a test sphere of a radius r and aconcentric shell of radius R=r+δ the sphere displaying probes at adensity σ˜P⁰/r². The effective probe concentration within the shell isgiven by

$\left\lbrack P_{s} \right\rbrack = {\frac{3{\sigma \cdot r^{2}}}{R^{3} - r^{3}}.}$

Letting R decrease toward r, that is, in the limit δ-->0, the localprobe density approaches the limit

${\left\lbrack P_{s} \right\rbrack = \frac{\sigma}{\delta}};$

in this limit, probes may be viewed as “condensed” on the bead surface.

For example, given a bead of diameter 3.2 μm and a typical value of P⁰of 10⁶ per bead, σ˜10 ⁵ μm⁻². The effective probe concentration within ashell of dimension δ=0.1 μm is thus:

[Ps]≈3×10⁵[μm⁻²]/0.1[μm]≈3×10⁶×10⁻²⁴ [M]/10⁻¹⁵[L]˜3 mM.

Typical conditions for target capture in solution involve a choice ofprobe concentration equal to the maximal anticipated targetconcentration. Assuming a dynamic range of 2 orders of magnitude, theprobe concentration will exceed the lowest detectable targetconcentration by not more than 2 orders of magnitude. Thus, in order topermit detection of target at a concentration of 10 nM (see Example 1),a typical probe concentration will be 1 μM. The effective probeconcentration associated with the bead thus exceeds, by at least 3orders of magnitude, that typically encountered in solution.Accordingly, as a target approaches the solid phase carrier surface, itencounters probes with a far higher probability than that governing suchencounters in solution, and this translates into a correspondinglyhigher local concentration of probe-target complexes. This inventiondiscloses, immediately, below, a hopping model permitting the target tointeract, during each encounter with the bead surface, with not one, butmultiple probes, thereby extending its residence time near the surface.

Enhanced Detection Sensitivity: Target “Hopping” andRecapture—Experimental observations, described in greater detail inExample 1 and in FIGS. 4 and 5, especially in the upper panel of FIG. 5,for a looped probe attached by its loop subsequence to a microparticle(“bead”), indicate the response to display, in the regime of low targetconcentration, a substantially enhanced detection sensitivity ascompared to the response of that probe in solution.

The enhancement is attributed to target “hopping” from occupied tonearby unoccupied capture probes (see FIG. 6A, B). That is, targetsexecute random walks (of varying extent) on the surface by hopping fromsite to (unoccupied) site. If “hopping” can occur sufficiently rapidlyso as to leave the target conformation essentially unchanged and thus“primed” for recapture (FIG. 6A), this process will increase theresidence time of the target at or near the surface. Denoting by T thecharacteristic relaxation time of the target conformation, from itsconstrained state it must adopt for association with thecarrier-displayed probe, to the unconstrained state it adopts as it“escapes” into the bulk solution (FIG. 6C), the distance, d_(NN),between any occupied probe site and the nearest unoccupied site(s) so asto permit (random) “hopping” on a timescale τ_(h)<τ. Denoting by μ_(h) acharacteristic hopping mobility, and corresponding diffusivityD_(h)=(kT/M)μ_(h), M representing the mass of the target molecule, thiscondition translates into d_(NN) ²<D_(h)τ or, for the probe density, aσ˜d_(NN) ⁻²>1/D_(h)τ.

Phenomenologically, the increase in target residence time manifestsitself in the form of a reduction in the observed rate of dissociation.The ratio, k_(d)/k_(d0), of the observed to the “intrinsic” ratedecreases with increasing probability of a target completing a “hop”from its current probe site to a nearby (unoccupied) probe site, andthis probability, Θ, in turn increases with the number of probes P⁰provided on the surface, and with the unoccupied fraction, 1−Γ, of thoseprobes. Thus, k_(d) may be represented in a form

k _(d) =k _(d0)└1−Θ(P ⁰,1−Γ)┘

where Θ(P⁰,1−Γ) represents the probability of target recapture at a siteclose to the site of release; Θ(P⁰,1−Γ) will be a monotonicallyincreasing function of P⁰ and 1−Γ, and max(Θ)≦1.

Solving for Γ. from the detailed balance equation, k_(a)(1−Γ)T_(s)=k_(d)Γ yields:

${\Gamma \equiv \frac{\lbrack{PT}\rbrack}{P^{0}}} = {K_{0}{T_{s}\left( {1 + {K_{0}T_{s}} - {\Theta \left( {P^{0},{1 - \Gamma}} \right)}} \right)}}$

where K₀=k_(a)/k_(d0) represents the affinity constant observed in theabsence of target retention; in the limit of low target concentration,or small affinity constant, Γ=KT_(s).The observed affinity constant,

K=K ₀[1−Θ(P⁰,1−Γ)]⁻¹.

is enhanced at low target concentration, reflecting the large fractionof capture sites available to each target molecule; K decreases towardits “intrinsic” value at high coverage. Regardless of its detailed form,the recapture probability function, Θ(P⁰,1−Γ), relates an increase inobserved affinity to an increase in total surface probe density and/ordecrease in coverage. By enhancing the observed affinity, thiscooperative effect arising from target hopping between densely graftedprobes on a solid surface favors complex formation and thus accounts foran enhanced sensitivity. The arguments advanced herein are not limitedto the self-complementary (“looped”) probes employed here, and willapply to any target (or ligand) capture to solid-phase displayed captureprobes (or receptors) at low target (or ligand) concentration.Interfacial Polarization—At high stringency, capture especially of shorttargets will occur within a polarized interfacial region of elevatedionic strength, and hence under conditions of lower stringency ascompared to conditions in the bulk solution. For example, for a 50-mMbulk NaCl concentration, this interfacial region extends to acharacteristic length 1/κ˜30 Å beyond the surface of the solid phasecarrier. Given the increased effective target concentration, this willfurther stabilize the OT state, a conclusion which also follows from theanalysis of the mathematical description described above (see Eq 1).Under these conditions, an effect such as a counterion-mediatedattraction of short range (Ha & Liu, Phys Rev Letts. 79, pp 1289-1292(1997)) may contribute to target retention within the interfacialregion.Expanded Dynamic Range—The experimental observations described in theExamples below also indicate the response of looped probes anchored to asolid surface to display a more than two-fold expansion of dynamic rangeas compared to that observed in solution.

At typical grafting densities of at least 10⁵ probes per bead, a solidphase assay, especially in the regime of low target concentration,corresponds to conditions of excess probe. Under the assumption, α˜γ,β˜0, discussed above, and under the further assumption K_(c)>>K_(o), theabsolute fluorescence intensity assumes the form:

F _(ab) =αP ⁰ T ⁰(P ⁰ +K _(c))⁻¹ =αT ⁰(1+K _(c) /P ⁰)

This expression, describes an increase in the intensity of fluorescenceemitted by looped probes with increasing probe density. That is, theresponse, given by the slope, α(1+K_(c)/P⁰)⁻¹, in fluorescence intensityas a function of variations in target concentration, will affect theintensity of emitted fluorescence. For example, under conditionsdescribed in Example 1, K_(c)≈0.1 μM, so that, if the grafting density,and hence P⁰ is varied from (an equivalent of) 10 nM to (the equivalentof) 10 mM, the response in fluorescence signal intensity can be variedover an order of magnitude, from 0.1α to α.

The broadening in the response is reminiscent of that observed whencomparing the response of a polyclonal antibody to that of a monoclonalantibody (Tarnok, Hambsch, Chen & Varro, Clinical Chemistry 49, No. 6,pp 1000-1002, 2003). However, as described herein, anchored loopedprobes, grafted at high density, also display an enhanced detectionsensitivity at low target concentration. This effect, which has not beendescribed in connection with immunoassay designs replacing a monoclonalcapture antibody by a polyclonal capture antibody, is attributed here toan enhanced observed (“effective”) affinity at low coverage inaccordance with a target hopping model.

In accordance with the target hopping model, a cooperative effectrelated to probe grafting density enhances the affinity observed at lowcoverage, thereby further contributing to the heterogeneity in theresponse in a manner that is favorable to generating an expanded dynamicrange of target detection. At low target concentration, the response isdominated by the enhanced affinity arising from target retention nearthe surface, and at high target concentration, the response is dominatedby the low affinity associated with low grafting density. That is, theexpanded dynamic range reflects the contributions of enhancedsensitivity at low coverage, and those of solid phase carriers of loweraffinity at high coverage.

2—Formation of eOT State: Enhancing Operating Range and DetectionSensitivity—

The use of a looped probe calls for operation within a range of optimalstringencies that is determined by a trade-off between detectionsensitivity and specificity. Conditions of low stringency will stabilizethe C state, thereby rendering target capture more difficult andreducing detection sensitivity. Conversely, conditions of highstringency will destabilize both the C state and the OT state, asevident from the results of the detailed mathematical descriptionprovided herein above, thereby reducing specificity: in the extreme, theopen state of the probe will produce fluorescence even in the absence oftarget.

Optimization of specificity generally will dictate selection of anoperating temperature near the melting temperature of the relevantprobe-target complex. However, as this choice also reduces the stabilityof the probe-target complex, it reduces detection sensitivity.Conversely, a choice of lower stringency increases the sensitivity, butcompromises the specificity of the response. When detection of target bycapture to looped probes is to be performed concurrently with enzymatictarget amplification (or other enzyme-catalyzed target manipulation) ina homogeneous format, or subsequent to such manipulations, but withoutintervening separation step, in a “single-tube” format, the choice ofoptimal stringencies may be further constrained. In practice, highstringency is preferred: for example, the conditions of Example 1,involving the formation of a duplex of 20 base pairs, provide for 50 mMsalt and an operating temperature of 42C.

Optimal stringencies generally will depend not only on specific captureprobe sequences, but on target configuration and/or length, and the taskof identifying the operating range of stringencies in a multiplexedassay thus becomes increasingly difficult, given the dispersion of themelting curve profiles of a set of different probe-target complexesunder given assay conditions. The design of a multiplexed assay formatcalling for the concurrent detection of multiple targets by capture tomatching probes, will thus further restrict the choice of optimalstringencies which depend on the stability of individual probe-targetcomplexes.

Thus, target-mediated elongation of (the 3′terminal subsequence of) aself-complementary probe provides a method of stabilizing probe-targetcomplexes by converting the OT state into the elongated (“eOT”) stateand thereby a method of expanding the operating range particularly ofmultiplexed nucleic acid detection while simultaneously enhancing thesensitivity of detection. Elongation may be performed using DNA targetand a DNA polymerase or RNA target and a Reverse Transcriptase (RT), asdescribed in the co-pending application included herein by reference.The probe is constructed so as eliminate “self-priming”, either byproviding strictly blunt ends of the stem, or preferably by providing an“overhanging” 3′terminus.

Expanding the Operating Range—The enhanced thermodynamic stability ofthe eOT state manifests itself in a shift to higher temperature of themelting curve: generally, the longer the template, the larger shift. Incontrast, since the 5′ terminal subsequence of the probe remainsunmodified, the C-->O transition follows its original melting curve. Ina multiplexed assay, this shift of the dispersive portion of the meltingcurves of different probe-target complexes to higher temperature,renders the system more forgiving in terms of selecting a high operatingtemperature: as illustrated in this situation FIG. 7A, the ability tooperate at high temperature ensures high stringency and hencespecificity, and the ability remain outside of the range of dispersionsimultaneously ensures high sensitivity. Non-uniform probe elongation,as a result of randomly aborted probe elongation reactions, wouldproduce a polydisperse length distribution and would further broadenthis distribution of affinity constants. Such an increase inheterogeneity will manifest itself in an increase in the dispersion ofthe (shifted) melting curves (see FIG. 7B); that is, randomly abortedelongation reactions provide a means of expanding the dynamic range ofthe assay.Enhancing the Sensitivity—The enhanced stability of the eOT state alsotranslates into enhanced detection sensitivity, as a result of shiftingthe equilibrium of the competitive probe-target interaction to theduplex state by converting OT states, essentially irreversibly, intostable eOT states. Phenomenologically, this conversion corresponds to aa reduction of the observed rate of dissociation, and correspondingincrease in the observed affinity of the probe-target interaction: tothe extent that it is irreversible, this process, given sufficient time,will consume all available target.

The enhancement in detection sensitivity afforded by generation of the(essentially irreversible) eOT state is particularly effective whenoperating in a regime of stringency permitting only the transientformation of an OT state. Random fluctuations producing the transientformation of a probe-target-enzyme-substrate intermediate will mediatethe (essentially) irreversible conversion of a fraction of thisintermediate OT state into an eOT state, leading, over time, toaccumulation of eOT state and depletion of target. The “zippering-up” ofthe intermediate OT state producing the eOT state, akin to the turn of aratchet, permit operation in a regime of low stringency without loss ofdetection sensitivity.

3—Allele-Specific Detection and Phasing

As with allele-specific detection of nucleic acids generally, loopedprobes may be used to advantage in connection with Elongation-mediatedMultiplexed Analysis of Polymorphisms (eMAP™; see U.S. application Ser.No. 10/271,602). In this application, the use of a looped probe has theadditional benefit of permitting control of molecular stringency so asto improve allele discrimination by target capture. In particular, eMAPusing looped capture probes which simultaneously serve as elongationprimers permit the application of phasing, either in the mode describedin detail in U.S. application Ser. No. 10/271,602 (incorporated byreference), or by combining the stringent control of annealingconditions afforded by the design of specific stem subsequences withallele-specific elongation of a 3′-terminal subsequence whose 3′terminus is designed not to display complementarity with the 5′-terminalsubsequence so as to eliminate the possibility of self-priming.

That is, as illustrated in FIG. 8, the configuration of a first variablesite, located within the portion of the sequence capable of annealing tothe 3′-terminal subsequence of the probe is detected by preferentialcapture of the matching allele, and the configuration of a secondvariable site, located in juxtaposition to the 3′terminus (or proximalposition) of the probe, is detected by elongation (or lack thereof).Elongation products may be formed under conditions permittingincorporation of fluorescently labeled dNTPs or may be formed withunlabeled dNTPs and decorated by a fluorescently labeled hybridizationprobe; such a decoration probe can be designed to be directed to anadditional polymorphic site of interest located in the elongated probesequence.

Example I Homogeneous Beadchip Assay Using Looped Probes

A homogenous BeadChip assay format, shown in FIG. 1, was implemented byproviding a variable gap configuration set to a large value duringtarget capture and a smaller value during recording of assay images froma random encoded array of beads displaying self-complementary probes aswell as positive and negative controls. The reaction volume was sealedby encapsulation of the reaction with mineral oil (from Sigma-Aldrich).

BeadChips were prepared to contain a random array composed of 4,000beads of four types of color-encoded microparticles (“beads”) on a375-μm thick <100> n-type Silicon substrate. Color-coding was achievedby staining the beads in accordance with a solvent tuning methoddescribed in U.S. application Ser. No. 10/348,165 (incorporated byreference). Stained beads were functionalized by covalent attachment ofstreptavidin to permit subsequent attachment of biotinylatedself-complementary (“looped”) probes, illustrated in FIG. 1.

One probe, displayed on one type of bead, contained a 20-nt capturesequences specific to a 20-mer single-stranded target; the other probecontained an unrelated 20-mer sequence. Three type of beads wererespectively functionalized with a target-specific (“matched”) probe, amismatched probe serving as a negative control, and a biotinylated andCy3-modified oligonucleotide (“A10”) serving as an intensity reference;a fourth type of bead, left un-functionalized, was added to dilute thearray composition. BeadChips were affixed to glass substrates using anepoxy adhesive (“Loctite”) and a polydimethylsiloxane (PDMS) spacer,either 400 μm or 1,000 μm in thickness, was cast; PDMS conforms well toflat surfaces and provides a reliable seal, given its negligible thermalexpansion up to 100° C. Two 400-μm spacers were placed adjacent to themounted BeadChip, and two 1000-μm spacers were placed next to the 400-μmspacers; a glass coverslip of 0.15 mm thickness was cut to fit theseparation of the 1000-μm spacers.

To perform the assay, 1.5-μl of reaction mix containing specific targetat a particular concentration was pipette-transferred to the chipsurface; the reaction volume was closed by fixing the coverslip via twoPDMS pads placed onto the 1,000-μm spacers, and transferring 5-μl ofmineral oil into the gap; capillary forces ensure that the oil quicklyencircles and isolates the reaction volume. After completion of thereaction, the coverslip was shifted so as to come to rest on the 400-μmspacers to form a 25-μm gap for optical interrogation.

The result of titrating a 20-mer RNA target on a Beadchip using thissetup is shown in FIG. 5A at a temperature of 42C and in FIG. 5B at twoadditional incubation temperatures, followed by imaging at roomtemperature. Fluorescence intensity readings, normalized using the A10fluorescence, are shown along with normalized data recorded from thesame assay performed in solution, using a fixed looped probeconcentration of 0.1 μM. Compared to the solution response, the reactionwith the bead-displayed probes displays a much broader detection dynamicrange of target (3 logs) and substantially enhanced sensitivity at lowtarget concentration.

Example 2 Homogenous Assay in Suspension of Encoded Beads

The looped-probe design also can be used in a homogenous format withencoded beads in suspension, as described in U.S. Pat. No. 6,251,691;U.S. application Ser. No. 10/204,799 (incorporated by reference). Asshown in FIG. 9, a reaction mixture in a sealed incubation chamber, orcartridge, may contain T7-tagged DNA template, components for in-vitrotranscription reaction such as a T7 RNA polymerase, well known in theart, and looped-probe functionalized color-coded beads, each colorcorresponding to a unique capture probe sequence. Preferably, encodedmagnetic beads are used (see U.S. application Ser. No. 11/218,838), anda random array of such beads is assembled in real time followingcompletion of the assay, as described in U.S. Pat. No. 6,251,691; U.S.application Ser. No. 10/204,799.

Two sets of magnetic beads (Spherotech, 4.10 μm in diameter, ρ˜1.13g/ml), one encoded with a green dye by solvent-tuning (REF—SolventTuning), the other left uncolored, are covalently functionalized withStrepavidin for attachment of a biotinlyated looped probe. One probe,displayed on the green beads, contains a 10-nt capture sequence specificto a 20-mer HIV single-stranded target; the other probe contains a 10-ntsequence unrelated to HIV. The looped probes are labeled with a Cy3fluorescence dye on the 5′-terminal subsequence and a Blackhole quencheron the 3% terminal subsequence Buffer containing all the reactioningredients is adjusted in density by properly mixing with 20% FicollPM70 separation medium (Amersham) in D2O (Aldrich, ρ˜1.18 g/ml, η˜10cp). The reaction suspension is then brought to 0.25% solid content.

In-vitro transcription is performed in the sealed chamber, or in asealed cartridge, containing suspended beads (see also the detaileddescriptions in the co-pending application included herein byreference). The reaction is initiated by raising the temperature to apredetermined value optimizing the efficiency of the T7 RNA polymerase;the “hot start” mechanism, well known in the art, also may be employedto initiate the reaction.

Real-time Array Assembly and Detection—The cartridge is placed into amagnetic field configuration designed to permit the formation of arandom array of beads. Beads are first magnetically trapped at thesemiconductor surface and the reaction buffer exchanged for assemblybuffer, previously disclosed, preferred for the subsequent step: an ACvoltage (typically <1Vpp, <1 kHz) is applied to the electrodes and aspot on the substrate, defined by an aperture in the projection optics,is illuminated (typically with a power of 30 mW/mm² generated by a12V/100W Halogen Lamp), and a converging electrokinetic flow directedtoward the illuminated spot is induced near the semiconductor surface.Under the influence of both electrokinetic and magnetic-dipole-repulsiveforces, beads gather in the illuminated region but remain separated fromeach other. Finally, beads are “annealed” into a dense-packed orderedplanar assembly. Images are then recorded with a CCD camera (Apogee).

In an alternative arrangement, the fluorescence signal associated withthe open state of the looped probe may be detected by inserting thereaction mix into a flow cytometer which also permits decoding of thebeads and hence determination of sequences corresponding to each assaysignal.

Example III Homogeneous Binding Assay in Suspension Using Looped ProbesImmobilized on Magnetic Beads

Looped probes were immobilized on color-encoded magnetic microparticles(“beads”) for use in a homogeneous binding assay. Briefly, magneticbeads of ˜4 micron diameter were synthesized by standard methods andcolor-encoded as set forth in U.S. application Ser. No. 10/348,165,incorporated by reference. Next, encoded beads were modified by covalentattachment of Neutravidin to epoxy groups on the beads to permit:attachment of a “perfect-match (PM)” biotinylated looped probe, a“no-match (NM)” biotinylated looped probe, and a biotinylated positivecontrol, in the form of a Cy3-labeled oligonucleotide.

As in the previous examples, looped-probes contain a donor dye and anacceptor dye at their respective 5′ and 3′ ends. Aliquots ofprobe-decorated, encoded magnetic beads were pooled in one test tube fordetermination of RNA target concentrations.

To determine the response of the probes, target RNAs were seriallydiluted (1:2) in reaction buffer (50 mM Tris (pH 8.0), 0.1 mM EDTA, 50mM NaCl, 0.2% Tween 20) and were then each incubated with an aliquot ofpooled magnetic beads in a test tube. Following incubation for 10 min atroom temperature, a 0.5 μl aliquot of each bead suspension wastransferred—without washing—into an incubation chamber on a siliconwafer (FIG. 11) for image acquisition.

Trapping of magnetic beads was realized in a magnetic trap shown in FIG.12A. This device comprises a bottom actuation element and a disposabletop element that may host a channel system or a static reactor. In thisexample, it hosted an incubation chamber, as shown in FIG. 12A, whichwas formed by sandwiching 0.5 μl bead suspension droplet between a solidsubstrate and a 0.2-mm glass cover slip with 100-μm separation, and thenby encapsulating the liquid phase with mineral oil. The magneticactuator consists of a magnetic core, a coil, and high-permeabilityalloy layers that tune the field flux. In this particular embodiment,the device generates a magnetic field that is localized in a ⅛″ circularregion. To form an array of magnetic beads, a typical current below 100mA was sufficient to generate a flux density gradient exceeding by morethan two orders of magnitude that of an untuned coil withoutsignificantly increasing the flux density (<200 Gauss). Illustrated inFIG. 12B is the computed field distribution of quantities relevant tomagneto-phoresis, namely, equipotential curves of −b², a quantityproportional to magneto-phoretic potential of an induced magnetic dipolemoment, and vectors of its gradient, which is proportional to therelevant force. The induced magnetic field induces the magnetic beads insuspension to migrate towards a substrate. Once in proximity to thesolid support, the beads interact with each other repulsively andreorganize into arrays in the reaction buffer. The beads are in a randomstate before the magnetic field is turned on.

In this experiment, following incubation, bead suspension from each tubewas transferred into the magnetic trap and, on activation, organizedinto arrays in accordance with the method described above. Opticalinterrogation was performed using fluorescence microscope (Nikon EclipseE800). Image snapshots were taken through different optical filters,which are bright field, Cy3 filter (F5, 500 ms), green filter (F5, 200ms), and blue field (F5, 150 ms), respectively. Images were processedusing a Matlab code. Each single bead was identified and itscorresponding Cy3 intensity was then registered to its blue intensity.In a “bead-map” (FIG. 13) plotted with Cy3 against blue, three clustersof beads can be seen and can be categorized to be B1, B2, B3, from leftto right, respectively. The Cy3 intensity of B2 cluster indicates themagnitude of RNA-binding to the looped probes of specific type. Afternormalizing to the positive control (B1) for each sample. The doseresponse of target interaction with specific and non-specific loopedprobes are summarized in FIG. 14, with error bars representing standarddeviation of the mean intensities.

It should be understood that the terms, expressions and examples hereinare exemplary only and not limiting, and that the scope of the inventionis defined only in the claims which follow, and includes all equivalentsof the subject matter of the claims.

1-3. (canceled)
 4. In a multiplexed assay method carried out insolution, wherein the solution contains nucleic acid targets and,wherein several different types of oligonucleotide probes, each typehaving a different sequence in a target binding domain, are bound to asubstrate and used to detect the nucleic acid targets, and wherein saidprobes have a target binding domain and a complementary closing domaincapable of forming a duplex with the target binding domain wherein whenthe duplex is formed, no signal is emitted by the probe, and a joiningregion between the target binding domain and the closing domain, andwherein the same signal is generated by a probe in a non-duplex as by aprobe bound to the target or by an elongated probe bound to the target,the method comprising: placing the probes in contact with the targetsunder conditions suitable for capture of the target and formation of aprobe-target duplex; generating conditions suitable for enzyme-mediatedprobe elongation wherein the 3′ terminal end of the probe is elongatedif a nucleotide in the target sequence which is aligned with the 3′terminal end of the target binding domain is complementary; anddetecting the increase in cumulative signal associated with each type ofprobe, resulting from probe elongation.
 5. The method of claim 4 whereinthe detection of elongation is by detecting a signal associated withlabeled nucleotides incorporated into the elongated probe.
 6. The methodof claim 5 wherein labeled dNTPs or ddNTPs are incorporated into theelongated probe.
 7. The method of claim 4 wherein detection ofelongation is performed by conducting a thermal stability analysis, bycycling to a temperature above the de-annealing temperature ofnon-elongated duplexes and then monitoring probe fluorescence todetermine probe-target-associated fluorescence.
 8. A method of expandingthe operating range of stringencies of a multiplexed format of nucleicacid analysis, wherein a solution contains nucleic acid targets and,wherein several different types of oligonucleotide probes, each typehaving a different sequence in a target binding domain, are bound to asubstrate and are used to detect the nucleic acid targets, and whereinsaid probes have a target binding domain and a complementary closingdomain capable of forming a duplex with the target binding domain,wherein when the duplex is formed, no signal is emitted by the probe,and a joining region between the target binding domain and the closingdomain, and wherein the same signal is generated by a probe in anon-duplex as by a probe bound to the target or by a probe bound to thetarget and elongated, the method comprising: stabilizing the duplex byelongating the 3′ terminal ends of certain probes which have anucleotide in the target sequence aligned with a complementarynucleotide in the target binding domain to thereby generating a stableduplex capable of withstanding a wider range of reaction conditionswithout causing changes in the assay results.
 9. A method of conductinga multiplexed format of nucleic acid analysis, wherein a solutioncontaining nucleic acid targets is placed in contact with severaldifferent types of oligonucleotide probes, each different type having adifferent sequence in a region designated as a target binding domain,wherein when the duplex is formed, no signal is emitted by the probe),and a joining region between the target binding domain and the closingdomain, and wherein the same signal is generated by a probe in anon-duplex as by a probe bound to the target, the method comprising:said target binding domain joined to a complementary closing domainthrough a joining region, the method comprising: adjusting assayconditions so as to permit stabilization of probe-target complexes bytarget-mediated enzymatic elongation; and detecting capture bymonitoring probe fluorescence from the target-associated state of theprobe and comparing it to the pre-assay signal.
 10. The method of claim9 further including increasing the effective concentration of thetargets available for binding to the probes by one or more of thefollowing: (i) adjusting the solution's ionic strength to greater than athreshold; (ii) selecting a target domain of a size less than athreshold; or (iii) selecting target domains within a specifiedproximity to a terminal end of the targets.
 11. The method of claim 8 or9 wherein the reaction time is selected to reflect the stringency of theimposed conditions, the stringency determining the probability of randomformation of a probe-target-enzyme-substrate intermediate state in theformation of elongation product, such that a sufficient number of stableelongated duplex states are capable of being formed.
 12. (canceled) 13.The method of any of claim 4, 8, or 9, wherein the substrate is amicroparticle.
 14. The method of any of claim 4, 8, or 9, wherein theionic strength of the solution is increased by adding salt.
 15. Themethod of any of claim 4, 8, or 9, wherein there are several differenttypes of oligonucleotide probes on each microparticle.
 16. The method ofany of claim 4, 8, or 9, wherein the target binding domain is fullycomplementary to the target domain.
 17. The method of any of claim 4, 8,or 9, wherein the oligonucleotide probes and the nucleic acid targetscan either be DNA or RNA.
 18. The method of claim 10, wherein the ionicstrength threshold corresponds to a buffer concentration of 50 mM salt.